Project Summary/Abstract I propose to investigate the regulation of morphogenesis using the Caenorahbditis elegans embryo as a model. Defects in morphogenesis lead to developmental abnormalities in humans, and morphogenetic models have provided insight into wound closure and human diseases, including cancer. The tenant of morphogenesis is that small cell shape changes and movements structure tissues to provide shape. The cytoskeleton and cell adhesion are instrumental to cell shape changes and movement, but their regulation is only partially understood. The Bowerman lab has generated a large collection of ~1000 temperature-sensitive, embryonic lethal (TS-EL) mutants that provide a rich resource for investigating embryonic morphogenesis. TS-EL mutants are useful because earlier essential requirements for a gene can be bypassed by growth at the permissive temperature, and upshifts to the restrictive temperature simultaneously reduce both maternal and zygotic gene function. Therefore, I will perform the first systematic and large-scale forward genetic screen for TS-EL, morphogenesis-defective mutants and characterize the morphogenetic requirements for the genes I identify. In aim 1, I will complete my classification of terminally differentiated mutant phenotypes, genetically characterize the mutations that result in early and penetrant morphogenesis defects, and identify the genes affected by these mutations. Thus far, I have identified mutations in 9 conserved genes and have chosen six for further analysis based on their conservation in humans and because their roles in morphogenesis are not known. In a collaboration with Dr. Zhirong Bao at Memorial Sloan Kettering Cancer Center, we are using a semi-automated approach to determine the cell fates produced during embryogenesis in these and other mutants. I will focus further effort on those mutants with normal cell fate patterning that are therefore specifically defective in morphogenesis. In aim 2, I will use fluorescent epidermal membrane markers to identify and sort into shared trait groups those mutants with defects in epidermal cell shape changes and movements that mediate early embryonic morphogenesis. For mutants with defects in these morphogenetic processes, I will determine where the wild-type genes are expressed by making fluorescent protein fusions at endogenous loci using CRISPR/Cas9. Our lineage analysis already has shown our TS-EL mutation in HLH-1, the body wall muscle master regulator, specifically affects only body wall muscle cell fate and positioning, suggesting a previously unknown influence of body wall muscle cells on the overlying epidermal cell dynamics that mediate morphogenesis. To further explore this intriguing result, I will use fluorescent fusion proteins and live imaging to determine if muscle cell fate or positioning defects correlate with defects in epidermal cell shape and position during early morphogenesis. My goal is to advance our understanding of the genetic pathways that regulate morphogenesis and in the longer term explore in more depth the requirements for individual genes that we identify and prioritize.